Examination of thymic cell types by multiparameter flow cytometric analysis of cell size and cell surface antigens has allowed the detection of seven subpopulations of mouse thymocytes. Three of the subpopulations have not been previously described. One was extensively characterized and was demonstrated to have the properties of an early precursor thymocyte. Thymic subpopulations have been isolated to higher purity using monoclonal antibodies to cell surface antigens using cytotoxic and panning methods. These subpopulations have been examined for proliferative capacity, morphology, steroid and radiation sensitivity, function, as well as differentiation potential. Isolated thymic subpopulations have been assayed for the expression of T cell antigen receptor at the mRNA and protein level. Cell fusions have been made between the isolated subpopulation of thymocyte thought to be early precursors and the BW cell line. These cell fusion products will be studied for T cell receptor gene rearrangements before and after induction of cell surface antigens with phorbol compounds and mitogens. Ontogenetic studies on the development of the fetal thymus have demonstrated a sequence of development of various cell types with characteristics similar to thymic cell types found in adult mice. Studies on the differentiation potential of the isolated putative early thymocytes both in vitro and in vivo have confirmed its precursor status and suggested a sequence of differentiation similar to that seen in fetal thymus. This system is being further dissected for numbers of precursors and lineage relationships. The relationship of these early thymocytes to prothymocytes originating in the bone marrow (BM) is under investigation.